Preparation and application of biological material for targeted therapy of osteoarthritis

Abstract

The invention discloses preparation and application of a biological material for targeted therapy of osteoarthritis, and the preparation method comprises the following steps: according to a phage screening principle, after phages are combined with cartilage cells, washing off phages which are not combined with the cartilage cells, are not firmly combined with the cartilage cells and are non-specifically combined with the cartilage cells; eluting the specifically bound bacteriophage with glycine acid; after the specific binding bacteriophage is amplified by escherichia coli, screening is carried out by utilizing a bacteriophage display technology and cartilage cells; the preparation method comprises the following steps: preparing a small-size porous microgel loaded drug by adopting a multiple Pickering emulsion template method; and grafting the screened cartilage targeting peptide to the surface of the porous hydrogel microsphere to obtain the HS-SP microsphere. According to the invention, the problem of OA targeting can be effectively solved by adopting a phage display technology, and a drug is precisely targeted and delivered to an OA lesion region; the porous hydrogel microspheres with small particle size are prepared by adopting a multiple Pickering emulsion method, so that the use of a large number of surfactants is avoided, and the efficient loading and release of drugs can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to the preparation and application of biomaterials for targeted treatment of osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a common chronic joint disease characterized by degenerative changes in articular cartilage, subchondral bone remodeling, synovitis, and osteophyte formation. It is the main cause of pain and physical disability in the aging population. As of 2020, there are 595 million OA patients worldwide. Symptoms include joint pain that develops gradually and worsens after activity, stiffness that lasts for <30 minutes when getting up in the morning or at rest, and occasional joint swelling. X-rays can assist in diagnosis, and treatment includes joint protection, muscle strengthening rehabilitation, and pain-relieving drugs. Existing biomaterials for targeted treatment of osteoarthritis generally use cartilage-targeted peptides, and existing cartilage-targeted peptides are mainly based on phage display technology. After binding to cartilage and removing the cartilage matrix, cartilage-targeted peptides are obtained; in addition, there are peptides that bind to chondrocyte-specific proteins through antibody-antigen binding.

[0003] For cartilage targeting peptides, there are currently only targeting peptides for cartilage matrix, and there are no targeting peptides for chondrocytes, OA cartilage, OA chondrocytes, and OA cartilage proteins. Therefore, although the current cartilage targeting peptides have a certain effect of targeting cartilage, their specificity with chondrocytes is not good, and they cannot bind to OA cartilage or OA chondrocytes, resulting in the inability to accurately deliver the drug to the OA site. At the same time, the loading efficiency of the carrier loaded with the drug is low, so it is difficult to accurately and efficiently treat OA. In addition, direct drug delivery will have problems such as large drug doses and easy removal by synovial fluid; therefore, a carrier is needed to protect the drug and play a role in sustained release. Hydrogel microspheres are widely used as drug carriers due to their biocompatibility, modifiability, and injectability; however, the most commonly used method for preparing hydrogel microspheres is microfluidics. Although microfluidics can obtain microspheres with a single and controllable particle size, their particle size is too large and difficult to inject, and it is difficult to have a porous structure, resulting in low drug loading efficiency. At the same time, a large amount of surfactants will be used in the preparation process, which is difficult to remove and reduces biocompatibility. Summary of the invention

[0004] The purpose of the present invention is to address the shortcomings of the prior art and to propose a biomaterial preparation and application for the targeted treatment of osteoarthritis. Through phage display technology, OA cartilage, OA chondrocytes and OA proteins are specifically bound, which can not only more accurately deliver drugs to the site of OA occurrence, but also use the multiple Pickering emulsion method to prepare porous hydrogel microspheres, which can efficiently load drugs and achieve accurate and effective drug treatment of OA.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing a biomaterial for targeted treatment of osteoarthritis comprises the following specific steps:

[0007] S1: According to the phage screening principle, after the phage binds to the chondrocytes, the phages that do not bind to the chondrocytes, bind weakly, or bind non-specifically are washed away;

[0008] S2: specifically bound phages were eluted by glycine;

[0009] S3: After the specific binding phages were amplified with E. coli, they were screened again with chondrocytes using phage display technology;

[0010] S3a: The “panning-elution-amplification” process was repeated three times to screen out phages that specifically bind to chondrocytes;

[0011] S3b: The specifically bound phages were subjected to ELISA affinity assessment and DNA sequencing;

[0012] S3c: The sequencing is translated into an amino acid sequence with a certain sequence, and the cartilage-targeting peptide SP is synthesized by solid phase according to the amino acid sequence;

[0013] S4: Preparation of small-sized porous microgels loaded with drugs using multiple Pickering emulsion templates;

[0014] S4a: HA, monomer SBMA, crosslinker Laponite and photoinitiator are dissolved in water as the continuous phase to obtain an O / W primary emulsion, and a small amount of liquid paraffin is used as the dispersed phase;

[0015] S4b: Liquid paraffin was added again as the continuous phase to prepare an O / W / O emulsion, which had an obvious multiphase morphology;

[0016] S5: The cartilage-targeting peptide SP screened by phage display technology in S3 was grafted onto the surface of the porous hydrogel microspheres HS prepared in S4 to obtain HS-SP microspheres.

[0017] As a further technical solution of the present invention, in S3, the phage display technology inserts the DNA sequence of the foreign protein or polypeptide into the appropriate position of the phage coat protein structural gene, so that the foreign gene is expressed along with the expression of the coat protein, and the foreign protein is displayed on the phage surface along with the reassembly of the phage.

[0018] As a further technical solution of the present invention, in S4, the small-sized porous microgel loaded with drugs prepared can achieve the purpose of injectability, high-efficiency loading and sustained release of drugs.

[0019] As a further technical solution of the present invention, in S4a, the crosslinking agent Laponite stabilizes the O / W primary emulsion, and the particle size thereof is uniform, and the particle size is about 10 μm.

[0020] As a further technical solution of the present invention, in S4b, under ultraviolet light, the SBMA monomers in the emulsion are polymerized and cross-linked to form porous hydrogel microspheres HS, and the particle size is uniform, about 20 μm.

[0021] As a further technical solution of the present invention, the porous hydrogel microspheres HS are micrometer-scale three-dimensional network structure spheres formed by cross-linking hydrophilic molecules.

[0022] The beneficial effects of the present invention are:

[0023] 1. Through phage display technology, specific targeting peptides of OA cartilage, OA chondrocytes, and OA proteins can be screened out, which can effectively solve the OA targeting problem. OA targets can be screened for OA cartilage, OA chondrocytes, and OA proteins, and the drugs can be precisely targeted and delivered to the OA lesion area.

[0024] 2. Multiple Pickering emulsions were used to prepare small and porous hydrogel microspheres, and the screened cartilage-targeting peptides were grafted onto the surface of the hydrogel microspheres. This not only avoided the use of a large amount of surfactants, but also achieved efficient loading, sustained release and targeted drug delivery, thereby realizing precise treatment of OA. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 (a) Schematic diagram of phage display technology; (b) DNA sequence inserted into blue plaques; (c) ELISA test of positive phages and chondrocytes; (d) Affinity of peptides and chondrocytes;

[0026] Figure 2 A flow chart of the preparation of a biomaterial for targeted treatment of osteoarthritis proposed by the present invention;

[0027] Figure 3 Multiple Pickering O / W / O emulsions and microspheres (a) and W / O emulsions and microspheres (b); (c) particle size distribution of HS microspheres prepared by O / W / O and W / O emulsions; SEM of HS microspheres prepared by O / W / O (d) and W / O (e) emulsions; (f) optical microscopy and fluorescence microscopy of HS-SP microspheres; (g) SEM and EDS of HS-SP microspheres. DETAILED DESCRIPTION

[0028] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0029] Example 1

[0030] Please see attached Figure 1 -Attached Figure 3 , a method for preparing a biomaterial for targeted treatment of osteoarthritis, comprising the following specific steps:

[0031] S1: According to the phage screening principle, after the phage binds to the chondrocytes, the phages that do not bind to the chondrocytes, bind weakly, or bind non-specifically are washed away;

[0032] S2: specifically bound phages were eluted by glycine;

[0033] S3: After the specific binding phages were amplified with E. coli, they were screened again with chondrocytes using phage display technology;

[0034] S3a: The “panning-elution-amplification” process was repeated three times to screen out phages that specifically bind to chondrocytes;

[0035] S3b: The specifically bound phages were subjected to ELISA affinity assessment and DNA sequencing;

[0036] S3c: The sequencing is translated into an amino acid sequence with a certain sequence, and the cartilage-targeting peptide SP is synthesized by solid phase according to the amino acid sequence;

[0037] According to the phage screening principle, after the phage binds to the chondrocytes, the phages that do not bind to the chondrocytes, bind loosely, or bind non-specifically are washed away. Then, the specifically bound phages are eluted with glycine. Finally, the specifically bound phages are amplified and then screened with the chondrocytes again ( Figure 1 a).

[0038] After three repeated rounds of screening, it can be seen that the amount of phage recovered in each round is increasing; the second round was enriched 20 times compared with the first round, indicating that phages specifically binding to chondrocytes were effectively enriched; compared with the second round of screening, the results of the third round of screening showed no obvious enrichment, indicating that the third round of screening tended to saturation.

[0039] After completing three rounds of screening, 20 blue plaques were randomly selected from a plate for amplification, and DNA was extracted for sequencing ( Figure 1b). Only 15 plaques obtained the inserted sequence. The amino acid sequence was deduced according to the brief genetic code table provided by the kit. Four amino acid sequences appeared in the 15 plaques (Table 1), among which the HPTISGPANPAFGGG sequence had the highest frequency, appearing 8 times. The affinity of the 15 blue plaques to chondrocytes was evaluated by ELISA experiments. The results showed that the affinity of the amino acid sequences HPTISGPANPAFGGG and SPSLIYFPKSIYGGG to chondrocytes was much higher than that of the other two amino acids ( Figure 1 c). Therefore, HPTISGPANPAFGGG and SPSLIYFPKSIYGGG amino acid sequences were selected for subsequent experiments.

[0040] Table 1 Selected amino acid sequences and frequencies of occurrence in blue plaques

[0041]

[0042] *: There is no corresponding insertion sequence in the corresponding DNA sequencing results.

[0043] In order to further evaluate the targeting specificity of the two targeting peptides HPTISGPANPAFGGG and SPSLIYFPKSIYGGG to chondrocytes. The two peptides were labeled with FITC and incubated with chondrocytes. The fluorescence intensity around the cells was observed under a fluorescence microscope to illustrate the targeting specificity of the peptides to chondrocytes ( Figure 1 d). Both HPTISGPANPAFGGG and SPSLIYFPKSIYGGG peptides are targeted to chondrocytes, but the targeting of SPSLIYFPKSIYGGG is significantly stronger than that of HPTISGPANPAFGGG. In addition, compared with the cartilage-targeted peptide CAP reported in the literature, the green fluorescence signal in the chondrocytes of the SPSLIYFPKSIYGGG group was higher than that of the CAP group. Semi-quantitative analysis of mean optical density further supported these findings, indicating that the chondrocyte uptake in the SPSLIYFPKSIYGGG group was significantly enhanced ( Figure 1 d). These results highlight the superior specific targeting effect of the SPSLIYFPKSIYGGG peptide fragment on chondrocytes. Therefore, the SPSLIYFPKSIYGGG peptide fragment was selected for subsequent experiments.

[0044] S4: Preparation of small-sized porous microgels loaded with drugs using multiple Pickering emulsion templates;

[0045] The multiple Pickering emulsion template method, also known as the multiple Pickering emulsion method, is an oil-in-water-in-oil emulsion stabilized by solid particles (such as colloidal silica, lithium terephthalate and other nanomaterials).

[0046] S4a: HA, monomer SBMA, crosslinker Laponite and photoinitiator are dissolved in water as the continuous phase to obtain an O / W primary emulsion, and a small amount of liquid paraffin is used as the dispersed phase;

[0047] S4b: Liquid paraffin was added again as the continuous phase to prepare an O / W / O emulsion, which had an obvious multiphase morphology;

[0048] The small-sized porous microgel loaded with miR-140 prepared by the multiple Pickering emulsion template method can achieve the purpose of injectable, efficient loading and sustained release of miR-140. First, HA, monomer SBMA, crosslinker Laponite and photoinitiator 2959 were dissolved in water as the continuous phase, and a small amount of liquid paraffin was used as the dispersed phase; Laponite stabilized the O / W primary emulsion, and its particle size was uniform, about 10μm; then, liquid paraffin was added again as the continuous phase to prepare the O / W / O emulsion, which had a clear multiphase morphology ( Figure 3 a); while directly mixing the oil phase and the water phase to prepare the W / O emulsion, the particle size is not uniform ( Figure 3 b); Under ultraviolet light, the SBMA monomers in the emulsion polymerize and cross-link to form hydrogel microspheres HS.

[0049] Optical microscopy, particle size distribution and SEM show that the HS microspheres prepared by O / W / O emulsion have obvious porous structure, and the particle size is uniform, about 20μm, and the distribution is narrow; while the HS microspheres obtained by W / O emulsion have no porous structure and the particle size is not uniform ( Figure 3 ce); then, the cartilage targeting peptide SP was grafted onto the surface of the microspheres to obtain HS-SP microspheres; by fluorescence microscopy and EDS, it can be seen that the SP peptide was successfully grafted onto the microspheres ( Figure 3 f,g).

[0050] S5: The cartilage-targeting peptide SP screened by phage display technology in S3 was grafted onto the surface of the porous hydrogel microspheres HS prepared in S4 to obtain HS-SP microspheres.

[0051] In a preferred embodiment, in S3, the phage display technology inserts the DNA sequence of the foreign protein or polypeptide into the appropriate position of the phage coat protein structural gene, so that the foreign gene is expressed along with the expression of the coat protein, and the foreign protein is displayed on the phage surface along with the reassembly of the phage.

[0052] The principle of phage display technology is as follows: the coding gene of the polypeptide or protein or the target gene fragment is cloned into the appropriate position of the structural gene of the phage coat protein, and the foreign polypeptide or protein is fused with the coat protein under the condition that the reading frame is correct and the normal function of other coat proteins is not affected. The fusion protein is displayed on the phage surface as the daughter phage is reassembled; the displayed polypeptide or protein can maintain a relatively independent spatial structure and biological activity, so as to facilitate the recognition and binding of the target molecule; after a certain period of incubation of the peptide library with the target protein molecule on the solid phase, the unbound free phage is washed away, and then the phage adsorbed and bound to the target molecule is eluted with a competitive receptor or acid, and the eluted phage infects the host cell and is multiplied and amplified, and the next round of elution is carried out. After 3 to 5 rounds of "adsorption-elution-amplification", the phage specifically bound to the target molecule is highly enriched, and the obtained phage preparation can be used to further enrich the target phage with the desired binding characteristics.

[0053] In a preferred embodiment, in S4, the prepared small-sized porous microgel loaded with drugs can achieve the purpose of injectability, high-efficiency loading and sustained release of drugs.

[0054] In a preferred embodiment, in S4a, the crosslinking agent Laponite stabilizes the O / W primary emulsion, and the particle size thereof is uniform, and the particle size is about 10 μm.

[0055] In a preferred embodiment, in S4b, under ultraviolet light, the SBMA monomers in the emulsion are polymerized and cross-linked to form porous hydrogel microspheres HS, and the particle size is uniform, about 20 μm.

[0056] In a preferred embodiment, the porous hydrogel microspheres HS are micrometer-scale three-dimensional network structure spheres formed by cross-linking hydrophilic molecules.

[0057] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: through the phage display technology, the specific targeting polypeptides of OA cartilage, OA chondrocytes, and OA proteins screened out can effectively solve the OA targeting problem. OA targets can be screened for OA cartilage, OA chondrocytes and OA proteins, and the drugs can be accurately targeted and delivered to the OA lesion area.

[0058] Multiple Pickering emulsions were used to prepare small and porous hydrogel microspheres, and the screened cartilage-targeting peptides were grafted onto the surface of the hydrogel microspheres. This not only avoided the use of a large amount of surfactants, but also achieved efficient loading, sustained release and targeted drug delivery, thereby realizing precise treatment of OA.

[0059] Example 2

[0060] A method for preparing a biomaterial for targeted treatment of osteoarthritis comprises the following specific steps:

[0061] S1: According to the phage screening principle, after the phage binds to the chondrocytes, the phages that do not bind to the chondrocytes, bind weakly, or bind non-specifically are washed away;

[0062] S2: specifically bound phages were eluted by glycine;

[0063] S3: After the specific binding phages were amplified with E. coli, they were screened again with chondrocytes using phage display technology;

[0064] S3a: The “panning-elution-amplification” process was repeated three times to screen out phages that specifically bind to chondrocytes;

[0065] S3b: The specifically bound phages were subjected to ELISA affinity assessment and DNA sequencing;

[0066] S3c: The sequencing is translated into an amino acid sequence with a certain sequence, and the cartilage-targeting peptide SP is synthesized by solid phase according to the amino acid sequence;

[0067] S4: Preparation of small-sized porous microgels loaded with drugs using multiple Pickering emulsion templates;

[0068] S4a: HA, monomer SBMA, crosslinker Laponite and photoinitiator are dissolved in water as the continuous phase to obtain an O / W primary emulsion, and a small amount of liquid paraffin is used as the dispersed phase;

[0069] S4b: Liquid paraffin was added again as the continuous phase to prepare an O / W / O emulsion, which had an obvious multiphase morphology;

[0070] S5: The cartilage-targeting peptide SP screened by phage display technology in S3 was grafted onto the surface of the porous hydrogel microspheres HS prepared in S4 to obtain HS-SP microspheres.

[0071] In the above steps, the small-sized porous microgel loaded with miR-140 prepared by the multiple Pickering emulsion template method can achieve the purpose of injectable, efficient loading and sustained release of miR-140. First, HA, monomer SBMA, crosslinker Laponite and photoinitiator 2959 are dissolved in water as the continuous phase, and a small amount of liquid paraffin is used as the dispersed phase; Laponite stabilizes the O / W primary emulsion, and its particle size is uniform, with a particle size of about 10μm; then, liquid paraffin is added again as the continuous phase to prepare the O / W / O emulsion, which has a clear multiphase morphology ( Figure 3 a); while directly mixing the oil phase and the water phase to prepare the W / O emulsion, the particle size is not uniform ( Figure 3 b); Under ultraviolet light, the SBMA monomers in the emulsion polymerize and cross-link to form hydrogel microspheres HS.

[0072] Optical microscopy, particle size distribution and SEM show that the HS microspheres prepared by O / W / O emulsion have obvious porous structure, and the particle size is uniform, about 20μm, and the distribution is narrow; while the HS microspheres obtained by W / O emulsion have no porous structure and the particle size is not uniform ( Figure 3 ce); then, the cartilage targeting peptide SP was grafted onto the surface of the microspheres to obtain HS-SP microspheres; by fluorescence microscopy and EDS, it can be seen that the SP peptide was successfully grafted onto the microspheres ( Figure 3 f, g);

[0073] The oil phase can also be replaced by a hydrophobic monomer, which includes: styrene, methacrylate, ethoxylated behenyl (meth)acrylate, ethoxylated tristyrylphenyl (meth)acrylate, ethoxylated hexadecyl (meth)acrylate, ethoxylated octadecyl (meth)acrylate, ethoxylated dodecyl (meth)acrylate and ethoxylated tetradecyl (meth)acrylate.

[0074] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0075] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a biomaterial for targeted treatment of osteoarthritis, characterized in that: The specific steps include: S1: According to the phage screening principle, after the phage binds to the chondrocytes, the phages that do not bind to the chondrocytes, bind weakly, or bind non-specifically are washed away; S2: specifically bound phages were eluted by glycine; S3: After the specific binding phages were amplified with E. coli, they were screened again with chondrocytes using phage display technology; S3a: The "panning-elution-amplification" process was repeated three times to screen out phages that specifically bind to chondrocytes; S3b: The specifically bound phages were subjected to ELISA affinity assessment and DNA sequencing; S3c: The sequencing is translated into an amino acid sequence with a certain sequence, and the cartilage-targeting peptide SP is synthesized by solid phase according to the amino acid sequence; S4: Preparation of small-sized porous microgels loaded with drugs using multiple Pickering emulsion templates; S4a: HA, monomer SBMA, crosslinker Laponite and photoinitiator are dissolved in water as the continuous phase to obtain an O / W primary emulsion, and a small amount of liquid paraffin is used as the dispersed phase; S4b: Liquid paraffin was added again as the continuous phase to prepare an O / W / O emulsion, which had an obvious multiphase morphology; S5: The cartilage-targeting peptide SP screened by phage display technology in S3 was grafted onto the surface of the porous hydrogel microspheres HS prepared in S4 to obtain HS-SP microspheres.

2. The method for preparing a biomaterial for targeted treatment of osteoarthritis according to claim 1, characterized in that: In S3, the phage display technology inserts the DNA sequence of foreign protein or polypeptide into the appropriate position of the phage coat protein structural gene, so that the foreign gene is expressed along with the expression of the coat protein, and the foreign protein is displayed on the phage surface along with the reassembly of the phage.

3. The method for preparing a biomaterial for targeted treatment of osteoarthritis according to claim 1, characterized in that: In S4, the prepared small-sized porous microgel loaded with drugs can achieve the purpose of injectability, high-efficiency loading and sustained release of drugs.

4. The method for preparing a biomaterial for targeted treatment of osteoarthritis according to claim 1, characterized in that: In the S4a, the crosslinking agent Laponite stabilizes the O / W primary emulsion, and the particle size thereof is uniform and about 10 μm.

5. The method for preparing a biomaterial for targeted treatment of osteoarthritis according to claim 1, characterized in that: In the S4b, under ultraviolet light, the SBMA monomers in the emulsion are polymerized and cross-linked to form porous hydrogel microspheres HS, and the particle size is uniform, about 20 μm.

6. The method for preparing a biomaterial for targeted treatment of osteoarthritis according to claim 1, characterized in that: The porous hydrogel microsphere HS is a micrometer-scale three-dimensional network structure sphere formed by cross-linking hydrophilic molecules.

7. An application of a biomaterial for targeted treatment of osteoarthritis, comprising the method for preparing a biomaterial for targeted treatment of osteoarthritis according to any one of claims 1 to 6.

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